Serum concentrations of BsAb-1 and BsAb-2 in mouse serum were determined using an antigen capture ELISA. All washes and dilutions were performed with freshly made TBS-T (Accuris). All volumes should be assumed to be 100 µL/well except for coating, blocking and washing, which are at 200 µL/well. The night before the assay, Nunc MaxiSorp™ flat-bottom plates (Invitrogen) were coated with recombinant human IL2Rγ protein diluted to 1 µg/mL in carbonate-bicarbonate buffer (Thermo Scientific) and left at 4 °C. The next day, the plates were washed 5 times and then blocked with 1% BSA for 30 min. The plates were washed once and then multiple dilutions of the serum samples were added, along with a reference standard. Stocks of known concentration for BsAb-1 and BsAb-2 were used to make the standard curve. After 1 h at room temp, the plate was washed 8 times and then biotinylated anti-human IgG4-Fc (MABTECH) diluted to 3 µg/mL was added. The plates were incubated at room temp for another 30 min and then washed again 8 times. Next, the plates were incubated for 30 min with HRP-Streptavidin (Thermo Scientific) diluted 1:4000. Following an additional 8 washes, the plates were incubated in the dark for 6 min with room-temperature 1-Step Ultra TMB (Thermo Scientific). The reaction was stopped with 100 µL/well 2 N sulfuric acid. Absorbance was assessed at 450 nm and 570 nm.
Nunc maxisorp flat bottom plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc MaxiSorp flat-bottom plates are a type of laboratory equipment designed for enzyme-linked immunosorbent assays (ELISA) and other similar applications. These plates feature a high-binding surface that enables effective immobilization of proteins, peptides, and other biomolecules. The flat-bottom design provides a consistent and uniform surface for reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (3 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Victor3 plate reader, by PerkinElmer (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Carbonate bicarbonate buffer, by Thermo Fisher Scientific (1 mentions)
1 step ultra tmb, by Thermo Fisher Scientific (1 mentions)
Hipo mpp 96 microplate photometer, by Biosan (1 mentions)
Igg2b, by Southern Biotech (1 mentions)
Igg2a, by Southern Biotech (1 mentions)
Uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Sandwich elisa kit, by BD (1 mentions)
Sandwich elisa kit, by Thermo Fisher Scientific (1 mentions)
Poly streptavidin hrp 20, by Biosynth (1 mentions)
Synergy 2 plate reader, by Agilent Technologies (1 mentions)
F ab 2 goat anti human iga hrp, by Thermo Fisher Scientific (1 mentions)
F ab 2 goat anti human igg hrp, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Goat anti rabbit hrp, by Promega (1 mentions)
27 protocols using nunc maxisorp flat bottom plate
1
Quantifying Bispecific Antibody Levels
2
Detecting Antibody Responses to HER2 and VAR2CSA
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96-well Nunc Maxisorp™ flat-bottom plates (Invitrogen, Carlsbad, CA, USA) were coated overnight at 4 °C with either recombinant HER2 (aa23-652, GenBank ID: NP_004439) or VAR2CSA (ID1-ID2a, FCR3 strain, GenBank ID: GU249598). HER2 coat protein was produced like its Catcher-counterpart [45 (link)], while VAR2CSA is described elsewhere [49 (link)]. Plates were blocked for one hour in 0.5% skim milk powder in PBS at room temperature (RT). Sera were diluted 100× and added to the well in threefold dilutions in blocking buffer and incubated for 1 h at RT. Plates were washed with 1xPBS three times in between steps. Plates were incubated for 1 h at RT with HRP-conjugated secondary antibodies targeting either total mouse IgG, IgG1, IgG2a or IgG2b (Invitrogen, Carlsbad, CA, USA). Plates were developed with TMB X-tra substrate (Kem-En-Tec, Taastrup, Denmark). OD450 was measured with a HiPo MPP-96 microplate photometer (Biosan, Riga, Latvia).
3
Quantification of Antibody Binding using ELISA
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Nunc MaxiSorp™ flat-bottom plates (Invitrogen™, Cat. No. 44-2402-21) were coated at 100 µL/well with one of the following: (1) 5 μg/mL DEX, (2) 5 μg/mL HSA, (3) 50 μg/mL P3DEX, (4) 100 μg/mL P3HSA, (5) 50 μg/mL NTDEX, (6) 100 μg/mL NTHSA, (7) 5 μg/mL SEA, or (8) 5 μg/mL SMP in 0.05 M carbonate bicarbonate buffer, pH 9.6, and incubated for 16 h at 4 °C. Plates were then washed 6× in PBS with 0.05% Tween-20 (PBST) and blocked via the addition of 300 µL/well of 2.5% milk in PBST at room temperature (RT) for 2 h. After additional washing, 100 µL/well of mice serum (1:100 in PBS) or hybridoma supernatant was added to the plate and incubated at RT for 2 h. Plates were washed and horseradish peroxidase-conjugated anti-mouse IgG (H+L) and IgM (Roche, Cat. No. 0311693001) or a panel of isotyping antibodies (IgG1, IgG2a, IgG2b, IgG3, and IgA; Southern Biotech, Birmingham, AL, USA, Cat. No. 5300-05) were added as required to the wells at a dilution of 1:4000 in PBST at RT for 1 h. Plates were washed four times, and color was developed using 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma, St. Louis, MO, USA, Cat. No. T0440). ELISA reactions were stopped after 10 min of incubation in the dark at RT with 50 µL of sulfuric acid. The optical density (OD) was determined using the SPECTROstar Nano Microplate Reader at 450/570 nm.
4
Cytokine Measurement via ELISA
5
Allergen-induced Immunological Responses
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Blood collected 24 h after allergen challenge was used for measuring the levels of rMet e 1-specific IgE, IgG1, and IgG2a at dilutions of 1:10, 1:10,000, and 1:200, respectively, on 96-well ELISA Nunc MaxiSorp flat-bottom plates (Invitrogen, Carlsbad, CA, USA), as described previously. Mouse mast cell protease-1 (mMCP-1) level was determined using sandwich ELISA kits (eBioscience, Santa Clara, CA, USA) as per the manufacturer’s instructions. Single cell suspension from individual spleens were prepared as previously described [53 (link)], and restimulated with 0.05 mg rMet e 1 at a density of 5 × 106 cells/mL for 72 h at 37 °C in a 5% CO2 incubator. Culture supernatants were then collected for the measurement of cytokine levels using sandwich ELISA kits (BD Biosciences, San Diego, CA, USA) in accordance with the manufacturer’s instructions.
6
Sandwich and Direct ELISA for Toxin Detection
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Sandwich ELISA’s were performed as previously described [54 (link)]. Briefly, 96-well black NUNC Maxisorp flat bottom plates (Thermo Scientific, Waltham, MA, USA) were coated with 100 µL/well of 1 g/mL of the capture antibody and incubated at 4 °C overnight. After overnight incubation the plates were washed two times in 0.02 M Tris buffered saline with 0.9% NaCl, pH 7.4, and 0.05% Tween-20 (TBST), blocked in 5% NFDM-TBST for an hour, and then incubated with samples in either PBS or LB at room temperature for 1 h. The plates were washed in TBST six times and incubated with the indicated detection antibody (100 ng/mL in NFDM-TBST). Goat anti-mouse HRP, or goat anti-rabbit HRP was added as a secondary antibody after washing six times in TBST. Plates were developed with 100 µL SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) and luminescence was read on a Victor3 plate reader (Perkin Elmer, Waltham, MA, USA) for 0.1 s. The limit of detection (LOD) was defined as the lowest toxin concentration at which the average ELISA reading was three standard deviations above the negative control. For direct ELISA’s, the plates were directly coated with 100 µL/well of 10 ng/mL of Stx2k toxin, followed by blocking, washing, and incubation with rabbit antisera at the indicated dilution and then goat-anti Rabbit-HRP as described above.
7
Quantifying Total and Oligomeric Amyloid-beta by ELISA
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The amount of total Aβ and oligomeric Aβ were evaluated by sandwich ELISA as described previously [27 (link)]. In brief, 100 μL of an anti-Aβ HJ3.4 (for oligomeric Aβ) or HJ2 (for total Aβ1–40) and HJ7.4 (for total Aβ1–42) antibody was coated to 96-well Nunc MaxiSorp flat-bottom plates (ThermoFisher) at 20 μg/mL in carbonate buffer overnight and then blocked with 2% BSA in PBS for 1 hour at room temperature. Samples and standard were loaded and incubated overnight; 6M guanidine-HCl was added at 5% of total sample volume for the total Aβ measurement to prevent oligomerization during the incubation. Biotinylated HJ3.4 antibody in PBS at 100 ng/mL was used as detection antibody and incubated at room temperature for 1 hour for the measurement of both total and oligomeric Aβ. Poly-streptavidin HRP-20 (Fitzgerald) in PBS at 30 ng/mL was then added and incubated for 30 min at room temperature. After final wash, the assay was developed by adding 100 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich) and the absorbance was read on a Synergy 2 plate reader (BioTek) at 650 nm.
8
ACPA Levels in Rheumatoid Arthritis Synovial Fluid
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ACPA IgA and IgG levels were determined in synovial fluids of RA patients according to the protocol previously described (20 (link)) with minor adaptations. Microtiter ELISA plates (Nunc MaxiSorp™ flat-bottom plates; ThermoFisher) were coated with 50 μl of 1 μg/ml streptavidin (ThermoFisher) and incubated overnight at 4°C after which 50 μl of 1 μg/ml biotinylated CCP2-cittruline or CCP2-arginine (kindly provided by Dr. J.W. Drijfhout, Department of Immunohematology and Blood Transfusion (Leiden University Medical Center)) was added for 1 hour at RT. Synovial fluid (1:10 diluted) was added for 1 hour at 37°C. Wells were washed and incubated with F(ab’)2 goat anti-human IgA-HRP (1:4000, ThermoFisher) or F(ab’)2 goat anti-human IgG-HRP (1:4000, ThermoFisher) for 1 hour at 37°C. Fifty μl of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added after which the reaction was stopped with 50 μl of sulfuric acid (10% H2SO4). Absorbance was measured with a microplate reader (Bio-Rad, Berkeley, CA) at 450nm.
9
Sandwich ELISA for Stx1-2 Detection
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Sandwich ELISAs were performed as previously described [23 (link)]. Briefly, 96-well black NUNC Maxisorp flat bottom plates (Thermo Scientific, Waltham, MA, USA) were coated with 100 µL/well of 1 µg/mL of the capture antibody, Stx1-2 [24 (link)] and incubated at 4 °C overnight. After overnight incubation, the plates were washed two times with 0.02 M Tris buffered saline with 0.9 % NaCl, pH 7.4, and 0.05% Tween-20 (TBST), blocked in 5% non-fat dry milk (NFDM)-TBST for an hour at 37 °C, and then incubated with samples for 1 h at 37 °C. The plates were washed with TBST six times and incubated with 100 ng/mL of detection antibody, Stx1 pAb [23 (link)], in NFDM-TBST. Goat anti-rabbit HRP (Promega, Madison, WI, USA), 10 ng/mL in NFDM-TBST, was added as a secondary antibody after washing six times with TBST. Plates were developed with 100 µL SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA), and luminescence counts per second (CPS) were read on a Victor 3 plate reader (Perkin Elmer, Shelton, CT, USA) for 0.1 s. Each treatment was performed in triplicate and repeated twice on two different days.
10
NK Cell Activation by SARS-CoV Spike Proteins
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PBMC from one control were rested overnight with 10 ng/ml human recombinant IL-15 (R&D Sytems) and NK cells were then isolated using EasySep Human NK Cell Isolation Kit (Stemcell Technologies). Nunc MaxiSorp Flat-Bottom plates (ThermoFisher) were coated with 1.5 ng/well of SARS-CoV-1 or SARS-CoV-2 spike proteins and incubated at 4 C for 6 h, then washed and blocked with R10 for 19 h at 4 C. After blocking, 100 ul of vNARs (5ug/ml) were added to each well and plates were incubated 30 min at 37 C. Plates were then washed with PBS 4x, followed by addition of 25,000 purified NK cells per well. GolgiStop (BD Biosciences) and Brefeldin A (ThermoFisher) were also added at 1:1000, and CD107a APC (clone H4A3; BD Biosciences) was added 1:100 in R10. After 6 h at 37 C, cells were stained for surface markers Aqua Live/Dead stain (ThermoFisher), CD3 PE-Tx Red (clone 7D6; ThermoFisher), CD56 PE-Cy7 (clone NCAM16.2; BD Biosciences), CD19 AF700 (clone HIB19; BD Biosciences), and CD16 BUV496 (clone 3G8; BD Biosciences) in FACS Buffer for 10 min at RT. Cells were fixed with Fix & Perm Medium A (ThermoFisher) for 15 m at RT. Fluorescence was evaluated on a LSRII (BD Biosciences). Data was analyzed using FlowJo version 10.7.1. NK cells were gated as CD3-CD19-/CD56+ CD16+, and CD107a expression was evaluated.
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